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T CELL HYBRIDOMA LYMPHOKINE CHEMOTACTIC FOR T LYMPHOCYTE

T 细胞杂交瘤淋巴细胞趋化因子

基本信息

批准号：
3445888
负责人：
JEFFREY H HILL
金额：
$ 5.63万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-07-01 至 1988-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3445888
关键词：
T lymphocyte antibody cell population study cell type chemoattractants chemotaxis electrofocusing flow cytometry gel electrophoresis helper T lymphocyte high performance liquid chromatography hybridomas radioimmunoassay suppressor T lymphocyte ultracentrifugation

项目摘要

Local T lymphocyte accumulation is thought to be important for tumor rejection and may presage local autoimmune disease and rejection of transplanted organs. The mechanisms of T lymphocyte accumultion at the site of initial antigen stimulation are not well defined, but it is known that antigen stimulated T cells produce a lymphokine(s) that is (are) chemotactic for other T cells. It is proposed that several different lymphokines that specifically attract either helper/inducer or suppressor/cytotoxic T cells may be released at different times and perhaps by different T lymphocyte subpopulations throughout the course of a local inflammatory response. Further, it is probable that these lymphokines play a significant role in determining the lymphocyte subpopulations that accumulate in a local inflammatory response and thereby, modulate the immune response. A T cell hybridoma, FS730.11, that produces large amounts of T cell specific lymphocyte chemotactic factor (LCF), but only a restricted range of other lymphokines has been developed. Con A stimulated supernate from cultures of FS730.11 will be used as a source of LCF from a single cell type to define whether helper or suppressor T cell subpopulations respond to this particular LCF. Helper and suppressor T cell subpopulations will be obtained by plating nylon wool non-adherent, mouse spleen cells on rat monoclonal anti-Lyt1 or Lyt2 antibody-coated plastic tissue culture plates. The purity of cell subpopulations will be assessed by flow cytrometry. Chemotaxis of these T cell subpopulations, in response to LCF, will then be assessed by migration through 8 micron pore size nitrocellulose filters in modified blindwell Boyden chambers. Semi-purified LCF will be injected into the peritoneum of mice. The peritoneal exudate cells will be quantified, differentiated by hematoxylin staining, and evaluated by flow cytometry for T lymphocyte. Purification of LCF will be initiated by sequential ultrafiltration and high performance liquid chromatography (HPLC). LCF will be characterized by sensitivity to inactivation with several enzymes, by disc gel electrophoresis, by isoelectric focusing and by column chromatography. Antibody to purified mouse LCF will be produced in rats. Intrinsically, radiolabelled LCF will be produced by Con A stimulation of FS730.11 in tissue culture, and a radioimmunoassay (RIA) for LCF developed. This RIA will facilitate future experiments to determine the T cell subpopulation(s) source of LCF and the kinetics of LCF production. A rat B cell hybridoma that secretes anti-LCF antibody will be produced, and antibody from that B cell hybridoma will be used in future experiments to study modulation of the immune response by inhibiting LCF activity in vivo.

局部 T 淋巴细胞积聚被认为对肿瘤很重要排斥反应，可能预示着局部自身免疫性疾病和排斥反应移植的器官。 T淋巴细胞聚集机制初始抗原刺激的位点尚不明确，但已知抗原刺激的 T 细胞产生一种或多种淋巴因子对其他 T 细胞具有趋化作用。提出了几种不同的淋巴因子，专门吸引辅助/诱导或抑制/细胞毒性 T 细胞可能会在不同的时间，可能是不同的 T 淋巴细胞亚群整个局部炎症反应的过程。进一步地，它是这些淋巴因子可能在决定在局部炎症反应中积累的淋巴细胞亚群从而调节免疫反应。 T 细胞杂交瘤 FS730.11，可产生大量 T 细胞特异性淋巴细胞趋化因子（LCF），但范围有限已开发出其他淋巴因子。 Con A 刺激上清液 FS730.11 培养物将用作单细胞 LCF 的来源类型来定义辅助性 T 细胞亚群或抑制性 T 细胞亚群是否有反应到这个特定的 LCF。辅助性和抑制性 T 细胞亚群将通过将尼龙毛非贴壁小鼠脾细胞铺在大鼠上获得单克隆抗 Lyt1 或 Lyt2 抗体包被的塑料组织培养物盘子。将通过流式评估细胞亚群的纯度细胞计数法。这些 T 细胞亚群响应 LCF 的趋化性，然后通过 8 微米孔径的迁移进行评估改良盲井博伊登室中的硝化纤维过滤器。将半纯化的LCF注射到小鼠的腹膜中。这腹膜渗出液细胞将被定量，并通过苏木精进行区分染色，并通过流式细胞术评估 T 淋巴细胞。 LCF 的纯化将通过连续超滤和高效液相色谱（HPLC）。 LCF将被表征通过对多种酶失活的敏感性，通过圆盘凝胶电泳、等电聚焦和柱色谱。纯化小鼠 LCF 的抗体将在大鼠体内产生。从本质上讲，放射性标记的 LCF 将通过 Con A 刺激 FS730.11 产生组织培养和 LCF 放射免疫分析 (RIA) 开发。本次RIA 将有助于未来确定 T 细胞亚群的实验 LCF 的来源和 LCF 生产的动力学。大鼠B细胞杂交瘤会产生分泌抗LCF抗体，并且来自该B的抗体细胞杂交瘤将在未来的实验中用于研究调节通过抑制体内LCF活性来调节免疫反应。